Peroxidases and Glycosidases in Intercellular Fluids from Noninoculated and Rust-Affected Wheat Leaves ¹: Isozyme Assay on Nitrocellulose Blots from Two-Dimensional Gels Free

Proteins in intercellular washing fluid (IWF) from noninoculated and stem rust-affected wheat leaves were separated by isoelectric focusing and polyacrylamide gel electrophoresis under nondenaturing conditions, transferred to nitrocellulose membranes, and assayed in situ for peroxidase and glycosidase activity.

Two infection-related peroxidase isozymes were detected in addition to more than ten that were present only in noninoculated plants. One other peroxidase isozyme was present in much higher concentration in IWF from infected leaves than in IWF from noninoculated leaves.

IWF contained many polymorphic glycosidases. A new method is described to localize the glycosidase isozymes accurately on nitrocellulose blots for evaluation of their substrate specificities: each blot was developed with the appropriate p-nitrophenyl glycoside to reveal glycosidase activity, then reprobed for concanavalin A-binding glycoproteins to serve as an internal reference frame for blot-to-blot comparisons. This procedure also provided information on glycosylation of the isozymes.

The locations of at least 15 (out of 37) isozymes were coincident with concanavalin A binding, including those of all 10 α-d-mannosidases, and of 6 β-d-xylosidases. On five areas of the blots there was coincidence of β-d-xylosidase and α-l-arabinosidase activity. Three of these areas corresponded to three of the most prominent Coomassie brilliant blue-stainable IWF proteins in gels. Isozymes that could convert p-nitrophenyl-β-d-glucoside, -β-d-galactoside, and/or -β-d-fucoside revealed a complex pattern of partially overlapping substrate specificities: two isozymes utilized both glucose and fucose derivatives, one utilized all three derivatives, and several others converted only one of the three substrates. No enzymes were detected with activity on p-nitrophenyl-β-d-galactosaminide, -β-l-fucoside, or -α-d-galactoside. No additional glycosidases were detected in IWF from stem rust-affected leaves.